Mesenchymal stem cells transfer mitochondria to the cells with virtually no mitochondrial function but not with pathogenic mtDNA mutations.

Department of Internal Medicine, Seoul National University College of Medicine, Seoul, South Korea.

Abstract

It has been reported that human mesenchymal stem cells (MSCs) can transfer mitochondria to the cells with severely compromised mitochondrial function. We tested whether the reported intercellular mitochondrial transfer could be replicated in different types of cells or under different experimental conditions, and tried to elucidate possible mechanism. Using biochemical selection methods, we found exponentially growing cells in restrictive media (uridine(-) and bromodeoxyuridine [BrdU](+)) during the coculture of MSCs (uridine-independent and BrdU-sensitive) and 143B-derived cells with severe mitochondrial dysfunction induced by either long-term ethidium bromide treatment or short-term rhodamine 6G (R6G) treatment (uridine-dependent but BrdU-resistant). The exponentially growing cells had nuclear DNA fingerprint patterns identical to 143B, and a sequence of mitochondrial DNA (mtDNA) identical to the MSCs. Since R6G causes rapid and irreversible damage to mitochondria without the removal of mtDNA, the mitochondrial function appears to be restored through a direct transfer of mitochondria rather than mtDNA alone. Conditioned media, which were prepared by treating mtDNA-less 143B ρ(0) cells under uridine-free condition, induced increased chemotaxis in MSC, which was also supported by transcriptome analysis. Cytochalasin B, an inhibitor of chemotaxis and cytoskeletal assembly, blocked mitochondrial transfer phenomenon in the above condition. However, we could not find any evidence of mitochondrial transfer to the cells harboring human pathogenic mtDNA mutations (A3243G mutation or 4,977 bp deletion). Thus, the mitochondrial transfer is limited to the condition of a near total absence of mitochondrial function. Elucidation of the mechanism of mitochondrial transfer will help us create a potential cell therapy-based mitochondrial restoration or mitochondrial gene therapy for human diseases caused by mitochondrial dysfunction.

Mitochondrial dysfunction was induced by long-term treatment of ethidium bromide (EtBr), which is known to remove mtDNA while sparing nuclear DNA, or by short-term treatment of rhodamine 6G (R6G), which tightly binds to the mitochondrial inner membrane and destroys the mitochondrial respiratory function. Gel image shows that the mtDNA was still present until five days after R6G treatment (inset). We cocultured MSCs with 143B-derived cells with severely compromised mitochondrial function (Δmt) in uridine− BrdU− media for five days (Stage I) and in uridine− BrdU+ media thereafter (Stage II). Cybrid cells harboring either the A3243G or 4,977 bp deletion mutation were also cultured in a similar way, with slight modification. For details, refer to the section. The gray-colored box refers to the condition, which was expected to be fatal for the cells.

(A) MSCs in DMEM had a fibroblast-like appearance. (B) MSCs survived in uridine-free media, (C) but they were sensitive to BrdU. (D) The 143B ρ0 cells in permissive media supplemented with uridine had an epithelial cell-like appearance. (E) The 143B ρ0 cells could not survive in uridine-free media, (F) but they were resistant to BrdU treatment. (G) At day 4 of Stage II coculture in uridine− BrdU+ media, fibroblast-like MSCs could not proliferate and gradually died with a senescent appearance. (H) After 7∼10 days in the uridine− BrdU+ media, we observed multiple foci of colonizing cells, which had distinct epithelial-like morphology of 143B cells on microscopic examination; (I) thereafter, they grew exponentially to reach confluence. All are magnified at ×40.

(A) The cells survived after coculture showed genetic identities with 143B ρ0 cell nuclei. The boxed numbers and corresponding peaks represent locations of polymorphisms for each short tandem-repeat marker. (B) Since ρ0 cells did not have any detectable mtDNA, the mtDNA of the cells survived after coculture came solely from the MSCs. The hypervariable region sequences of the cells were identical with the MSCs. The arrows indicate sequence variations compared to the Cambridge reference sequence. (C) PCR-RFLP for 10394 Dde I and 10397 Alu I of mtDNA. The recuperated cells in the coculture experiment with R6G treatment to a cybrid harboring mtDNA with +10394 Dde I and +10397 Alu I were revealed to have mtDNAs with both −10394 Dde I and −10397 Alu I, both of which were identical to the MSCs.

A scanning electron microscopic examination showed a marked difference in the morphology of the MSCs in each conditioned medium (magnified at ×1,000; scale bars represent 10 µm). The MSCs in their own growth medium (A); after 24 h (B) and 48 h (C) in conditioned media, which were prepared by treating 143B ρ0 cells with uridine-free media for 48 h. The MSCs showed an increased migration in the conditioned media without uridine supplementation (D). A transmission electron microscopic examination was done after two days of Stage I coculture (magnified at ×9,000; scale bars represent 1 µm). There were intercellular contacts between ρ0 and the MSCs by either cytoplasmic process (E) or broad surface contact (F).